Platelet Phosphoproteomic Profile Behind Anti-thrombotic Effects of Atypical Chemokine Receptor ACKR3

Introduction: Platelet hyperresponsiveness is linked to various cardio-/cerebrovascular, (immuno)thrombotic and thrombo-inflammatory diseases, requiring antiplatelet therapy. ACKR3 (formerly CXCR7), a noncanonical GPCR, exerts an antithrombotic, thrombo-inflammatory effect against arterial and venous thrombosis, following myocardial infarction and HIT. Pharmacological ACKR3-agonist alters the platelet lipidome limiting generation of prothrombotic but favoring antiplatelet lipids (12-HETrE) that upon release, engage G_αs-coupled canonical IP-receptor to trigger platelet inhibitory signalling involving Adenylyl Cyclase-cAMP-Protein Kinase A. However, the intricate phosphoproteomic signaling downstream of ACKR3 remained unexplored and is being addressed herein using time-resolved phosphoproteomics.

Method: We conducted (I) a temporal phosphoproteome analysis of ACKR3-agonist-stimulated resting platelets across seven time points (10-1800 s), and (II) a focused analysis on three key time points (early-10s, intermediate-600s, late-1800s), removing temporal effects in comparison to vehicle control-(DMSO)-treated platelets to ascertain ACKR3-specific effects ([Fig. 1]).

Fig. 1 Time-resolved Phosphoproteomics Analysis workflow Overview of the phosphoproteomics pipeline (left). Normalization method based on empirical phosphopeptides succesfully removes the batch effect caused by different donors. Our method combines thresholds on statistical significance and differential abundance to identify phosphopeptides that are stable throughout time and condition. We then remove the donor-associated variance by normalizing against the empirical phosphopeptides (right).

Results: We observed distinct phosphorylation profiles at early (10s,30s,60s) and late timepoints (300s,600s,900s,1800s) and a temporal change in the phosphoproteome of resting platelets, driven by CDK1, CDK5, PRKACA, PRKCA, PRKG1, and PRKAA1 signaling. Isolating the ACKR3 effect from the temporal changes highlighted ACKR3-specific signaling, with early downregulation of LCK and subsequent increases in SRC, LCK, and PKG activities. Key pathways affected included MET, ERBB2, mTOR, NO-GC-cGMP signaling, and clathrin-mediated endocytosis, suggesting significant modulation of tyrosine kinase receptor signalling, lipid synthesis, and endocytosis. Differentially regulated phosphosite-targets included HSPB1-S82 (PKA target), SRC-S17 (PKA target), BIN2-S282|S285 (predicted PKA target), PDE5A-S86, IRAG1-S189|S193 from cGMP signaling, SHC-S139, PTPN18-T393, and SRC-S17 from ERBB2 signalling networks. From a functional perspective and expanding on the previously reported IP-G_αs-AC-cAMP-PKA signaling triggered by ACKR3-agonist, we observed an upregulation in intraplatelet cGMP levels, and phosphorylation of PKA-PKG targets (pVASP-S157, pVASP-S239, PDE3A-S312, ENSA-S62/67, AMPK-T172, ACC1-S79) by immunoblots. We validated the involvement of AC-cAMP-PKA, GC-cGMP-PKG pathways in mediating the antithrombotic actions of ACKR3-agonist using pharmacological inhibitors of adenylyl-, guanylyl cyclase, PKA-PKG and sGC^-/- mice, which reduced the inhibitory effect of ACKR3-agonist on degranulation, aggregation and thrombus formation.

Conclusion: is the first study elucidating the complex temporal dynamics of signaling following a chemokine receptor (ACKR3) stimulation and validating its potential therapeutic efficacy to trigger the platelet inhibitory signaling cascade involving PKA-PKG.

Publikationsverlauf

Artikel online veröffentlicht:
13. Februar 2025